Seafood is a whale of an industry throughout the world.
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Explore This IssueDecember/January 2020
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Fish consumption grew from 19.8 pounds per capita in 1961 to 44.5 pounds in 2015, at an average rate of about 1.5 percent per year, according to a 2018 report from the Food and Agriculture Organization of the United Nations (FAO). Preliminary estimates for 2016 and 2017 point to further growth to about 44.75 pounds and 45.2 pounds, respectively, FAO projects.
Estimated U.S. per capita consumption of fish and shellfish was 16.0 pounds in 2017, an increase of 1.1 pounds from the 14.9 pounds consumed in 2016, according to the National Oceanic and Atmospheric Administration (NOAA).
In 2019 the FDA published its Evaluation of the Seafood HACCP Program for Fiscal Years 2006-2014. Presenting data from actual inspections, the report states, “The success rates for having and implementing HACCP (Hazard Analysis and Critical Control Points) plan controls for the hazards of pathogen growth/toxin formation and scombrotoxin were noticeably less than those for the other hazards.”
Prominent Seafood Pathogens
Foodborne pathogens typically associated with seafood products and seafood processing plant environments include Vibrio vulnificus, Vibrio parahaemolyticus, Salmonella spp., and Listeria monocytogenes, according to Kitiya Vongkamjan, PhD, an assistant professor in the Department of Food Technology at Prince of Songkla University in Hat Yai, Thailand.
Dr. Vongkamjan says the various rapid technologies available for detection of these pathogens offer several benefits, including:
- determination of specific pathogens in raw materials, finished products, and environmental samples
- detection of low numbers of pathogens in complex matrices of organic materials that are loaded with non-pathogenic microorganisms
- monitoring of process control, cleaning and hygienic practices during manufacture
- time, labor, and expense savings
“In contrast to conventional methods, rapid detection enables generation of fast and reliable results, which is especially important in light of ever-increasing global seafood trade requiring rapid transport over vast distances,” Dr. Vongkamjan relates.
Rapid detection methods can be categorized into nucleic acid-based, antigen-antibody based, biosensor-based, and phage-based methods, Dr. Vongkamjan notes.
Nucleic Acid-based Methods. Scientists have developed nucleic acid-based methods for detection and identification of specific DNA or RNA sequence of the target pathogen, Dr. Vongkamjan says. “Detection of a target nucleic acid sequence is performed by simple polymerase chain reaction (PCR), hybridization probes, or primers,” she elaborates. “Nucleic-acid based methods detect specific genes in the target pathogens associated with seafood.”
PCR-based methods are often classified into conventional PCR and real-time/quantitative PCR (qPCR), Dr. Vongkamjan explains. “Real-time PCR combines the specificity of conventional PCR with the quantitative measurement of fluorescence for monitoring amplification of specific genes in the target pathogens,” she explains. “A number of qPCR schemes have been designed to detect target genes such as the cholera toxin gene (ctxA) of V. cholerae or the tdh/trh genes of V. parahaemolyticus in fish and crustacean samples. Detection of multiple target genes of different species, serotype, or subtypes can be done in a single reaction by multiplex assay.”
Loop-mediated isothermal amplification (LAMP) is another variant of nucleic acid-based methods, Dr. Vongkamjan continues. “Most LAMP-based assays have been used for detection of V. parahaemolyticus, V. vulnificus, Salmonella spp., and L. monocytogenes in seafood and environmental samples. LAMP is proven to be more specific and sensitive compared to the other PCR-based assays for the detection of foodborne pathogens.”
Antibody-based Methods. Antibody-based detection relies on a highly specific and sensitive antibody-based system for the antigen present on the target pathogen, Dr. Vongkamjan says. “Most antigens contain amino acid sequences that are distinguishable among the target pathogens and other related non-target organisms,” she relates. “This specificity allows strong reactivity of antibody to the antigen in the target pathogen. Enzyme-linked immunosorbent assay is one such standard pathogen detection tool, whose detection system is based on enzyme-labeled reagents.”
Phage-based Detection Systems. Phages are viruses that can infect bacteria, Dr. Vongkamjan notes. “Wide-range applications of phages have been reported, including as pathogen detection systems,” she says. “Phages are typically modified to carry a gene such as a luciferase that encodes a protein, allowing for its rapid or easy detection. A real-time light emission produced by luciferase in the infected pathogen, such as Listeria, can be detected.”
Biosensor-based Methods. Biosensors are devices used to detect biological analytes, such as pathogens, according to Jane Ru Choi, PhD, a postdoctoral fellow in biomedical engineering at the University of British Columbia, Vancouver, Canada.
“These devices are named based on their detection approaches, such as colorimetric, fluorescent, electrochemical, and chemiluminescent-based biosensors,” Dr. Choi relates. “Biosensors can be implemented in point-of-care (POC) devices, which are diagnostic tools used to obtain results quickly close to the subject of the test.
With advances in POC testing, scientists have developed microfluidic chip-based devices including paper-based devices, such as lateral flow test strips and three-dimensional paper-based microfluidic devices, Dr. Choi says. Both microfluidic chip-based and paper-based devices can employ colorimetric, fluorescent, chemiluminescent, and chemiluminescent-based approaches, she elaborates.
“Despite some limitations, including poor sensitivity and lack of quantification, these emerging technologies are fast gaining popularity for use in detecting food contaminants, including those in seafood,” Dr. Choi points out. “POCs offer numerous advantages, including being affordable, sensitive, specific, user-friendly, rapid and robust, equipment free, and deliverable to end users.”
Certified Laboratories, Inc., based in Melville, N.Y., typically uses ribotyping for pathogen “fingerprinting” in seafood, according to Martin Mitchell, Certified’s chairman emeritus.
“Ribotyping is a molecular technique that capitalizes on unique genomic structures to differentiate strains of the pathogen,” Mitchell relates. “Ribotyping offers the benefits of molecular biology at less cost than whole-genome sequencing (WGS).
“Ribotyping and WGS refer to two specific techniques that fall under the broader term of “strain typing,” Mitchell continues. “Strain typing is any technique used to differentiate or determine the commonality of one strain of organism from another.”
According to Mitchell, strain typing is a useful tool for environmental monitoring in seafood processing establishments. “If, for example, sanitation is not effective in removing Listeria from a plant, strain typing can be used to determine if the organism came in on raw product, or if there are harborage issues in the facility,” he explains. “If the specific Listeria organism identified after sanitation is the same as the one identified before sanitation, that’s an indication there is a harborage issue.”
Supporting the U.S. Seafood Inspection Program
Fish and seafood products testing is conducted by the National Seafood Inspection Laboratory (NSIL), Pascagoula, Miss., to support the U.S. Department of Commerce Seafood Inspection Program through the NOAA National Marine Fisheries Service (NMFS), according to Jon Bell, PhD, NSIL director. “NOAA’s Office of International Affairs and Seafood Inspection (OIASI) is the U.S. competent export certification authority for fish and fisheries products,” Dr. Bell relates.
The NSIL supports the Seafood Commerce and Certification Division of the OIASI by verifying that U.S. exports meet importing governments’ food safety requirements, Dr. Bell notes. The verification process includes pathogen and indicator organism testing, along with processing audits conducted by the OIASI Seafood Inspection Program (SIP), he says.
“We conduct microbiological analyses on fish and fishery products for human consumption and aquatic fisheries byproducts to be used as ingredients in animal feeds and pet foods,” Dr. Bell elaborates. “We test for a number of microbiological contaminants, including Listeria, Salmonella, Staphylococcus aureus, fecal coliforms, and Vibrio bacteria, among others.”
“We also test for hazards in finished ready-to-eat seafood products, including cooked, packaged, vacuum packed, and frozen items,” adds Angela Ruple, MS, NSIL supervising lead analyst.
Ruple says NSIL laboratory professionals employ both traditional culture and more automated rapid methods, including PCR. “When testing products for compliance with SIP procedures requirements and export certification, we use validated methods from AOAC International and the FDA’s Bacteriological Analytical Manual,” she notes. “Since NSIL is International Organization for Standardization (ISO) 17025 accredited, we also do in-house validations and verifications.”
Molluscan Safety Issues
Dr. Bell notes that marine biotoxins are a growing concern within the Interstate Shellfish Sanitation Conference (ISSC), a cooperative body that implements the FDA’s National Shellfish Sanitation Program (NSSP). (He serves as the NOAA representative on the ISSC.)
“FDA regulates seafood safety, but states have overlapping responsibility through their public health programs and laboratories,” Dr. Bell points out. “Another established safety concern of the NSSP is naturally occurring Vibrios in harvest waters and shellfish.”
Dr. Bell says the NSIL supports Vibrio projects, including ecoforecasting by NOAA’s Coastal Ocean Services. “Ecoforecasting predicts how ecological events can indicate conditions that may impact human health, food, water, and the environment,” he explains.
Mollusk Safety: UK Focus
“When we talk about seafood safety at the Centre for the Environment, Fisheries and Aquaculture Science (Cefas), we generally mean safety of bivalve mollusks, oysters, mussels, and clams,” says Rachel Hartnell, PhD, principal scientist for seafood safety at Cefas.
An agency of the U.K. government’s Department of Food, Environment and Rural Affairs, Cefas operates two laboratories, one in Weymouth, Dorset, and the other in Lowestoft, Suffolk.
Cefas is the UK’s National Reference Laboratory for monitoring bacteriological and viral contamination of bivalve mollusks, and is responsible for coordination of the UK’s food safety official control program, with thousands of samples passing through the laboratories annually, Dr. Hartnell reports.
In February 2019, FAO designated Cefas as a Reference Centre for Bivalve Molluscs (European spelling) Sanitation. “This is the first time the FAO designated a Reference Centre in the mollusk sector,” says Dr. Hartnell, who serves as the Cefas lead for the center. “The mission of the center is to support the FAO vision for a globally unified system for shellfish safety.”
International collaborations are underway at the FAO Reference Centre. For example, in May 2019, the Cefas laboratory in Weymouth hosted the Joint FAO/World Health Organization (WHO) Expert Meeting on Microbiological Risk Assessment to update FAO/WHO guidance to reduce public health risks from pathogenic marine Vibrios.
At that meeting, 19 experts in the fields of genomics, epidemiology, risk assessment, pathogen detection, method standardization, and remote sensing from 13 countries focused their attention on how state-of-the-art methods could be used to inform risk assessments, Dr. Hartnell reports. “The long-range goal is the development of future international seafood safety standards,” she explains.
Commercial International Testing Services
NSF International is headquartered in Ann Arbor, Mich., but the presence and scope of its seafood services are worldwide.
“We provide seafood services from offices and labs in Everett, Wash.; Elizabeth, N.J.; Santiago, Chile; San Miguel, Peru; Guayaquil, Ecuador; Shanghai, China; Busan, South Korea; Delhi, India; Bangkok, Thailand; and Ho Chi Minh City, Vietnam,” says Tom White, global manager for certification and audits for NSF International’s seafood services.
NSF conducts full microbiological testing for seafood, including pathogens (Listeria, Salmonella, and E. coli O157:H7), standard plate counts, Coliform/E. coli, and yeast/mold, White notes. “For pathogens we run PCR analysis with culture confirmation,” he says.
In 2018, NSF conducted roughly 1,200 lab tests on seafood products, White reports. Testing environmental swabs from seafood processing facilities is another NSF service, he adds.
“The majority of our work is mainly with seafood processors, but we support a wide variety of clients, including canners and fishermen,” White relates.
“With 80 percent of all seafood consumed in the U.S. being imported from other countries, microbiological testing will continue to be an important step in protecting consumers from foodborne illnesses now and into the future,” White predicts.
Commercial PCR Test Kits for Seafood Safety
BIOTECON Diagnostics GmbH, Potsdam, Germany, offers a number of test kits for pathogens identification in fish and seafood products, according to Olaf Degen, MBA, the firm’s head of operational marketing.
The most recent offering, foodproof Listeria plus L. monocytogenes Detection LyoKit, introduced in 2019, is relevant for use with products like tuna salad and tuna sandwiches, Degen relates. “This LyoKit enables the simultaneous detection of the food-relevant sensu stricto Listeria species, L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, L. ivanovii, and L. marthii, as well as the specific identification of the pathogenic species, L. monocytogenes, in a single PCR reaction,” he elaborates.
The BIOTECON Diagnostics portfolio contains the foodproof Vibrio Detection LyoKit, which Degen says detects and differentiates between V. parahaemolyticus, V. vulnificus, and V. cholerae in a single PCR test. The Vibrio kit is particularly useful for quality control laboratories testing raw, ready-to-eat seafood and for shrimp aquaculture, Degen points out.
BIOTECON Diagnostics also offers the foodproof Norovirus (GI, GII) plus Hepatitis A Virus Detection Kit. “This specifically detects human pathogenic noroviruses of the genogroups I and II, and hepatitis A virus of the genotypes 1, 2, and 3 in a real-time PCR multiplex assay,” Degen says. “The virus test system has been validated with various matrices, including fresh oysters, mussels, shrimp, fresh and frozen tuna, sushi, and water.”
All of the BIOTECON Diagnostics pathogen test kits allow analysis to be performed in less than 24 hours with high sensitivity and 100 percent specificity, Degen notes. “In addition, yeast and molds can be detected in fewer than six hours directly from seafood samples using our foodproof Yeast and Mold Quantification LyoKit, instead of the usual five days it takes with standard methods,” he says.